1. Field of the Invention
The present invention relates to aggregation and dispersion methods of magnetic particles, separation and detection methods using the same and a detection kit.
2. Background Art
As a method of collecting a substance to be detected (also referred to as a target substance) in a mixed liquid (sample), a method in which microparticles on which a ligand capable of specifically adsorbing a substance to be detected has been immobilized are added to a sample thereby adsorbing the substance to be detected on the microparticles, thereafter, the microparticles are collected, and the substance to be detected is separated and collected from the microparticles is known. In particular, magnetic particles have a feature that they can be easily collected by a magnet, therefore, they are used as a means for efficiently collecting a substance to be detected. When the particle size of magnetic particles is more than 500 nm, the magnetic particles are easy to be magnetically collected, however, the adsorption reaction rate between the ligand on the surface of magnetic particles and the substance to be detected is not sufficient. On the contrary, when the particle size is 200 nm or less, though the adsorption reaction rate is high, the magnetic particles are difficult to be magnetically collected, and thus the substance to be detected cannot be collected.
Appl. Microbiol. Biotechnol., vol. 41, pp. 99-105, 1994 (Non-patent document 1) and Journal of Fermentation and Bioengineering, vol. 84, pp. 337-341, 1997 (Non-patent document 2) disclose stimulus-responsive magnetic particles (magnetic particles having a surface modified with a stimulus-responsive polymer) obtained by subjecting magnetic particles having a particle size of about 100 to 200 nm to surface modification with polyisopropyl acrylamide having a lower critical solution temperature (hereinafter sometimes referred to as “LCST”).
The magnetic particles having a surface modified with a stimulus-responsive polymer have a particle size of about 100 to 200 nm and are very small, therefore they are well dispersed in water, but cannot be magnetically collected in a dispersed state. However, in the case where an aqueous solution of magnetic particles having a surface modified with a thermoresponsive polymer is heated and the temperature thereof is raised to a temperature not lower than the LCST, the magnetic particles having a surface modified with a thermoresponsive polymer are deposited and aggregated. Since this aggregate can be easily collected by a magnetic force, an attempt has been made to perform separation of various biomolecules or microorganisms in a sample using the magnetic particles having a surface modified with the thermoresponsive polymer and is immobilized with an antibody or an antigen (an adsorbent).
However, in the case where collection is carried out using such a method, a heating and cooling system is required for raising the temperature of a sample containing the adsorbent using a magnetic particle having a surface modified with a thermoresponsive polymer to a temperature not lower than the LCST by heating thereby to aggregate or magnetically collect the adsorbent. Further, in the case where the adsorbent is applied to an immunodiagnostic system or the like for measuring a large variety of analytical items in a short time, a heating and cooling operation is required, therefore, the procedure is complicated. Further, in order to further reduce the time for analysis and measurement, a method of more promptly performing redispersion after once aggregating the adsorbent has also been demanded.